Studies have revealed that a major percentage of the red cells in all patients with sickle cell anemia (SCA) is ultimately transformed in vivo into permanently deformed irreversibly sickled cells (ISCs). However, the processes which cause this transformation remain unidentified. ISCs are rigid sickle cells which retain the sickled morphology even when oxygenated and devoid of polymerized hemoglobin-SS fibers. ISCs can constitute 2-30% of the red cells circulating in patients with SCA . The clinical complications associated with SCA are attributed to erythrocyte sickling and to the pathologic function of ISCs. The proposed investigation is designed to identify and characterize the mechanism(s) responsible for the transformation of sickle cells into the ISC morphology. Specifically, our studies will determine whether the mechanism involves membrane changes produced by accelerated red cell aging. Studies will determine correlations between age-related protein reorganization in the membrane of sickle cells and the generation of the ISC morphology. Experiments will detect and monitor age- related changes in the membrane of normal and sickle cells aged in vivo; and, aged in vitro under conditions which simulate natural aging processes. Based on our previous findings, we postulate that protein reorganization in sickle cells generates age-related cell surface antigens. Therefore age-related membrane changes will be probed experimentally by detecting the presence (or absence) of age-related cell antigen; and by studying changes in surface distribution and specificity of antigenic proteins. These data will be correlated with relative cell age and; changes in cell morphology and cell density which approach the properties identified for native ISCs. A major goal is to clearly define biochemical conditions of in vitro aging which produce permanent reorganization of membrane proteins in the chronologically young sickle cell. Antibody bound to cell antigens will be quantified by cytofluorography and Protein-A assays. Additional studies will determine if specific age-related protein reorganization occurs in sickle cells and whether this protein reorganization generates cell surface antigens unique to sickle cells. Longer term goals include studies of the influence of potential antisickling agents on membrane alterations produced by aging in vitro.